CN1798832A - Cultured cell construct containing spheroids of cultured animal cells and utilization thereof - Google Patents
Cultured cell construct containing spheroids of cultured animal cells and utilization thereof Download PDFInfo
- Publication number
- CN1798832A CN1798832A CNA028148428A CN02814842A CN1798832A CN 1798832 A CN1798832 A CN 1798832A CN A028148428 A CNA028148428 A CN A028148428A CN 02814842 A CN02814842 A CN 02814842A CN 1798832 A CN1798832 A CN 1798832A
- Authority
- CN
- China
- Prior art keywords
- cell
- constitutes
- spherule
- culturing
- thing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
- C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
- C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2535/00—Supports or coatings for cell culture characterised by topography
- C12N2535/10—Patterned coating
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
A culture containing patterned cultured parenchymal cell spheroids which can be formed by culturing parenchymal cells on cultured endothelial cells or cultured fibroblasts which have been separated by a specific hydrophilic polymer surface and patterned. This culture can sustain a function specific to the parenchymal cells over a long period of time.
Description
Technical field
The present invention relates to the culture of zooblast and the formation thing of this culturing cell, more particularly, relate to coculture and the surface of containing the culturing cell spherule and carry the biological device of holding this coculture.Biological device or coculture itself can be used for technical fields such as the inspection of animal cytophysiology for example or physiopathology situation or transplantation treatment, neomorph engineering science, hybridization type artificial organs.
Background technology
From functional point of view, zooblast roughly can be divided into the nonparenchymal cell (nonparenchymal cell) beyond parenchyma (parenchymal cell) and the parenchyma.Wherein, parenchyma is a cell of administering the function of tissue or organ.For example, the parenchyma of liver, promptly liver cell (hepatocyte) is born synthesizing, decompose, preserving of diversified material, is extremely important fundamental unit to life entity.
Thereby, for the expression of hepatocyte function in the imitative life entity of organism external mold, the various culture systems of this cell have been proposed.For example, United States Patent (USP) the 5th at R.Singhvi etc., 976, in No. 826 specification sheetss, put down in writing and cultivated hepatocellular method by cellular affinity (cytophilic) zone of the zone isolation that forms by wetting ability and the non-affinity of cell (cytophobic) material or be used for the device of this method.In this device, each cell inoculation water repellent surface or by have electric charge part (COO-,-PO
3On the surf zone of formation such as the constitutive protein matter of compound H-) or extracellular matrix (being generally 1-2500 μ m2, preferred 1-500 μ m2).In this patent specification, the cultivation of mainly putting down in writing or meaning to do can be thought and belongs to so-called monolayer culture.On the other hand, also attempted to improve hepatocellular function, the secretion that for example strengthens liver-specific protein matter be the stereoscopic culture of purpose (three-dimensionalgrowth pattern) (M.Smalley etc., In Vitro Cell.Dev.Biol.Anim. (1999) 35, pp.22-32).In this cultural method, on the gel of type i collagen or special extracellular matrix, cultivate the cell strain that derives from normal human liver cell.
In addition, as the device that can both use well based on the various purposes of toxicity test to each culturing cell basis of various parenchymas or nonparenchymal cell, also provide and the culturing cell patterning has been made it the device arranged by certain way [for example the spy opens flat 3-7576 number (apparatus is controlled in the arrangement with the cell on cell cohesiveness surface and cell non-adhesive surface), above-mentioned United States Patent (USP) the 5th, 976, No. 826 (hepatocellular cultivation), patent No. 2973976 (cultivation of endotheliocyte), spy open flat 7-308186 number (cultivation of endotheliocyte) etc.].
As mentioned above, compare with monolayer culture, in general stereoscopic culture can access have with organism in the culturing cell of the more approaching function of hepatocellular function, but the culturing cell of this stereoscopic culture system is peeled off from cultivating support easily, still on patterned surfaces, do not realize the example of stereoscopic culture system, with regard to holding time of the performance degree of above-mentioned functions and function etc., can't be met up to now.
In addition, the organ of original animal is that the tissue (set with cell of said function) by various character constitutes, and in most of the cases keeps its function etc. by the intercellular interaction of identical or different kind as the cell of its component unit.Therefore, at the The FASEB Journal of S.N.Bhatia etc., Vol.13 (1999); Among the 1883-1900, put down in writing based on and the nonparenchymal cell of parenchyma adjacency between heterocyst interaction pair cell propagation, move and/or viewpoint that differentiation is modified, to preserving the result that hepatocellular phenotype is studied with fibroblastic cultivate altogether (cocultivation) as nonparenchymal cell by liver cell.Point out in so common cultivation, if by cultivating nonparenchymal cell at grade, besieged on every side monolayer culture liver cell leaves 3 to 4 in its boundary line more than the cell, and then for example intracellular albumin generation ability is reduced to and the pure identical level of liver cell culture thing.Therefore, in this co-culture system, the cell of cultivating in the hepatocellular condensation product is difficult to all play consistently the liver specificity function.
Therefore, the object of the present invention is to provide a kind of specific function that comprises hepatocellular parenchyma stronger, and can long term maintenance, and the cultivation zooblast system that this pattern can stably be kept when carrying out micro-patterning (micropatterning).
Disclosure of the Invention
The inventor studies to the hepatocellular culture system one of in two kinds of mutually different cells with by the hepatocellular function of cultivation that this culture system obtains.Found that, if not according to above-mentioned S.N.Bhatia etc. pointed carry out the common cultivation of liver cell and nonparenchymal cell like that at grade, make the interaction of its generation by special-shaped interface (heterotypicinterface), but liver cell is cultivated on the cell monolayer of cultivating endotheliocyte or fibroblastic certain zone, then cultivating liver cell contacts with this individual layer surface, form the agglutinating spherule, and the spherule that forms like this has specific function to liver cell, for example can produce albumin steadily in the long term.In addition, the coculture that comprises the individual layer of these spherules and the cell different with the liver cell that forms the lower floor contact with these spherules; And the culturing cell that comprises this coculture and support thereof constitutes thing (below be sometimes referred to as spherule/culturing cell individual layer/support) and shows resistivity (stability) to peel off various culturing cells from support.Also find, when such culturing cell constitutes thing and is applied in parenchyma that can form spherule beyond the liver cell or the nonparenchymal cell, also can provide the culturing cell formation that contains the spherule that keeps these cell functions thing.
The inventor also finds, it is strong to form endotheliocyte in the cell of this spherule lower floor (or becoming the basis) or the general proliferative ability of inoblast and/or mobility, the tendency of emigration and propagation around the zonule of the support of oriented inoculation and cultivation, but, if this zonule is surrounded with the polymer surfaces that certain wetting ability is arranged and can become the non-affinity substance of cell, then can suppress culturing cells such as this endotheliocyte or inoblast from this zonule emigration or mobile, and can be suppressed at the emigration of the spherule that forms on these culturing cells or move, the coculture of such formation specific form can stably be remained on the support.
Therefore, according to the present invention, the culturing cell of coculture more than a kind or 2 kinds that contains different mutually zooblasts on the body that provides support constitutes thing.Each coculture that this culturing cell constitutes in the thing comprises:
A kind of in the mutually different cells preferably derives from the spherule of the culturing cell of parenchyma, and
Form lower floor contact with this spherule, and can make the substantial individual layer of the culturing cell formation of deriving from of the cells survival that forms this spherule and performance function other the a kind cell different with this cell.
As another program of the present invention, provide the biological device that comprises the surface that above-mentioned coculture forms more than 2 kinds.
As other scheme of the present invention, provide a kind of culturing cell to constitute the making method of thing, it is the making method that constitutes thing at the culturing cell of coculture more than a kind or 2 kinds that contains different mutually zooblasts on the support, this culturing cell constitutes the coculture that thing is included in the spherule that is loaded with culturing cell formation on a plurality of isolated zonules, it is characterized in that, on the material surface of coculture that should bond, formation is the polymer based layer with the polyoxyethylene glycol fragment, segmental any one the terminal covalent attachment of this polyoxyethylene glycol or not in conjunction with from constitute to the part of zooblast surface receptor in conjunction with the compound of selecting the monose in territory or oligosaccharides and the oligopeptides, by the covering pattern that a plurality of circular ports are arranged above-mentioned polymer layer is carried out Cement Composite Treated by Plasma, interval between each circular port is at least about 100 μ m, the diameter of circular port is about 50-100 μ m, remove polymer layer like this with corresponding zone, above-mentioned hole, in case of necessity, coating cellular affinity material in the zonule of having removed polymer layer, and in this zonule, cultivate endotheliocyte or inoblast, form the individual layer of culturing cell; Then, by on this culturing cell, cultivating the cell different, form the spherule of this difference cell with this culturing cell.
In addition, the present invention also provides the coculture itself that strips down from the support of above-mentioned culturing cell formation thing.
According to these the present invention, adopt above-mentioned liver cell and endotheliocyte or inoblast, reach and the same effect of cell that obtains by cultivation.For example, according to cultivation liver cell spherule of the present invention, minimum 3 weeks can be kept albumin high-levelly and be produced ability.
Description of drawings
Fig. 1 replaces representing that the glass surface according to hiding figure formation for preparing among the embodiment exposes the microphotograph of the accompanying drawing in hole.
Fig. 2 replaces the microphotograph of expression from the accompanying drawing of cultivating endotheliocyte bonding state on the cell cultures bed in the graphical hole of Fig. 1.
Fig. 3 replaces only bonding and form the microphotograph of accompanying drawing of the state of liver cell spherule on the diagram shape of Fig. 2 forms in conjunction with the endotheliocyte in territory of expression liver cell.
Fig. 4 is when replacing distance between the expression 100 μ m circular ports less than 100 μ m, the microphotograph of the accompanying drawing the when figure of cultivating endotheliocyte connects together.
Fig. 5 is the confocal some laser microscope photo that substitution list is shown in the accompanying drawing of the liver spherule 3 dimension images that diagram shape forms on the endotheliocyte.
The best mode that carries out an invention
According to the present invention, one of mutual different zooblast of cultivating altogether, so long as the cell by back described cultivation altogether can formation spherule can be any cell, preferred parenchyma.So-called parenchyma is meant in tissue or organ, becomes the cell of the part of bearing its function.For example, the parenchyma of liver is a liver cell, and the parenchyma of lung is an alveolar epithelial cells.Any cell that this definition comprised, follow the object of the invention all is the said parenchyma of the present invention.To the parenchyma indefinite, but, can exemplify liver cell, pancreas β cell, myocardial cell, neurogliocyte, skin epithelial cell, chondrocyte, osteocyte and embryo or adult stem cell as the parenchyma that lay special stress among the present invention will use.But,, be included in the above-mentioned zooblast even as long as the nonparenchymal cell outside the parenchyma that above-mentioned definition comprises as mentioned above, can form spherule, follow purpose of the present invention.
Another kind of cell as the mutual different zooblast of cultivating altogether, so long as when cultivating altogether, can play the cell that is different from this cell of the effect that makes above-mentioned cells survival that forms spherule and performance function, can be any cell, can exemplify cells such as endotheliocyte, epithelial cell, inoblast.Preferred endotheliocyte, particularly vascular endothelial cell, preferred especially umbilical cord vein blood vessel endotheliocyte.In addition, as other preferred cells, inoblast is arranged, inoblast is to disperse and be present in the animal individual mesoderm in nearly all tissue by leaf cell, according to using by carrying out the cell that above-mentioned cultivation forms spherule, that is the preferred form of the internal organs that this cell is existed of using forms the inoblast that plays an important role.In addition, to the combination of the mutual different zooblast cultivated altogether without limits, except aforesaid combination, also can be the nonparenchymal cell-nonparenchymal cell of inoblast-endotheliocyte, epithelial cell-endotheliocyte and so on; The combination of parenchyma-parenchyma of myocardial cell-liver cell, pancreas β cell-liver cell and so on.
Above cell for example, can be poultry, Mammals, the particularly cell in people source so long as the cell of the animal origin of its existence can be the cell of any animal.
When these mutual different zooblasts carry out common cultivation according to the present invention, endotheliocyte, epithelial cell or inoblast form on the surface of cultivating support and come down to the individual layer of culturing cell as support dependent cell or feeder cell, then, on this individual layer, cultivate with the different cell of cell that forms this individual layer, parenchyma preferably, contact with the culturing cell that constitutes this individual layer, interact, keep corresponding cell-specific function steadily in the long term by carrying out abnormal shape.Here the individual layer that said formation comes down to culturing cell be meant more than 80% of zone that forms this cellular layer, preferred more than 90%, more preferably constitute by the individual layer of culturing cell more than 95%.In addition, so-called cultivation altogether, can be in office phase when, above-mentioned different zooblast is mutually cultivated together, among the present invention, reference example is as at first cultivating endotheliocyte or inoblast, form individual layer after, the situation that these culturing cells are further cultivated with parenchyma then is more readily understood.
On the zonule of cultivating the support surface, cultivate for example endotheliocyte, epithelial cell or inoblast like this, on this zonule, be built into the feeder layer that these culturing cells form, then, the culturing cell that can be provided on the surface of feeder layer the coculture that constitutes with spherule that this feeder layer containing of directly contacting preferably derives from parenchyma constitutes thing.What is called derives from the spherule of the culturing cell formation of certain cell, and as the connotation of " spherule " this term, expression forms the aggegation piece that globular is cultivated parenchyma in fact; Be meant the form of being seen as in the additional confocal some laser microscope photo of Fig. 5, or form proximate with it.Be called and be spherical in fact, be not limited to spherically completely, also comprise the form that a flats is arranged, " being spherical in fact " is exactly to use under such intention.
As long as above-mentioned zonule can reach purpose of the present invention, then unqualified, generally can be to have about 1, about 200, the 000 μ m2 of 000-, preferred about 1, the Any shape of about 50, the 000 μ m2 of 500-, for example circular, based on the polygon of quadrangle, ellipse etc.Circular, at this moment, its diameter is the about 500 μ m of about 40-, preferred about 50-200 μ m, more preferably from about the about 100 μ m of 50-have on the larger-diameter zonule, feeder layer and the culturing cell formation thing that further is loaded with spherule in the above, show various cultures or coculture easily from the tendency of support sur-face peeling, the situation that spherule can not be stablized the function that keeps its origin parenchyma perhaps occurs.
As mentioned above, for example, on such zonule, when cultivating endotheliocyte, epithelial cell or inoblast, these culturing cell emigrations can not form culture on above-mentioned specific zonule to the zonule sometimes.
According to the present invention,, provide surface on every side, this zonule of encirclement made from the material of wetting ability and the non-affinity of cell as the means of avoiding this inappropriate situation.The details of relevant this material is described in the back, in the past, according to No. the 5th, 976,826, above-mentioned United States Patent (USP), the combination (to mediate cell binding) of prompting sialic acid, lectin, poly-semi-lactosi and other carbohydrate mediated cells; In addition, according to P.H.Weigel etc., J.Bio.Chem.Vol.254 (1979) 10830-10838, proved that for example liver cell is connected with carbohydrate specificity on covalentlying bind in smooth polyacrylamide gel, but said among the present invention " the non-affinity substance of cell " is independent mutually with these promptings, must note comprising on the contrary sometimes opposed material.
Specifically, the material of wetting ability of the present invention and the non-affinity of cell, be the material that can prepare surface: under the described in the back culture condition with following character, the culturing cell of above-mentioned support dependency culturing cell or formation feeder layer, can not be bonded in fully on the surface that this material makes, even perhaps bond, wash or do the wash also and can easily come off through demulcent, when cultivating parenchyma subsequently, can not stably carry out long-term cultivation, perhaps cultivate parenchyma and also be difficult to bonding.The material that a part can form this surface can be non-affinity of being put down in writing in the 5th, 976, No. 826 specification sheetss of above-mentioned United States Patent (USP) of formation cell or the material of dredging the unimolecular layer of biological (biophobic).As preferred material, can exemplify the compound that comprises polyoxyethylene glycol (hereinafter referred to as " PEG ") part.In addition, in this patent specification, specifically used compound (for example, the HS (CH that contains 6 ethylene glycol unit
2)
11(OCH
2CH
2)
6OH).
Further can preferably use in the present invention, and make the present invention become more unique be constitute the pair cell surface receptor part in conjunction with the territory, more particularly, although say liver cell and carbohydrate bonding, any terminal covalent attachment monose or oligosaccharides or certain oligomeric peptide can be used as the non-affinity substance use of cell based on the sugar derivatives of polyoxyethylene glycol fragment or peptide derivant or polysaccharide.
Therefore, the compound of the 5th, 976, No. 826 records of United States Patent (USP) also can be used in the surface that surrounds above-mentioned zonule, be polymer based but preferably use with the PEG segment.So-called is that the basis is meant and contains the PEG segment with the PEG segment, thereby do not have at these polymkeric substance under the situation of saccharide residue, formed surface is mainly covered by the free chain of PEG segmental, as long as can bring into play this effect, can be homopolymer or segmented copolymer, or their derivative.In addition, so-called free chain is meant when being placed in the aqueous medium that this segment can keep the state of the polymer chain of free conformation in fact.
Unqualified to this, this base polymer, the peptide derivant that further has the sugar derivatives of saccharide residue or have an oligomeric peptide residue can be represented with following general formula (I).
General formula (I)
Y-L
1-(B) m-L
2-(CH
2CH
2O) n-L
3In-X (I) following formula, L
1, L
2And L
3Represent atomic bond, Sauerstoffatom or joint independently, wherein m is 0 o'clock, L
1And L
2Can connect together, become atomic bond, Sauerstoffatom or 1 joint.
B represents following formula
Or
Wherein, R1 and R2 represent the alkyl of hydrogen atom, a 1-5 carbon atom independently, and p is the integer of 2-5;
X represents hydrogen atom, saccharide residue or peptide residue;
Y represent hydrogen atom or can with this polymkeric substance in conjunction with or adhere to the functional group of apparatus surface;
M is 0-10,000 integer, and
N is 0-20,000 integer.
In addition, L
1, L
2And L
3In, L
1The expression atomic bond ,-(CH
2) q-O-,-(CH
2) q-COO-,-(CH
2) q-S-or-CO-(CH
2) joint of q-NH-; Perhaps m is 0 occasion, L
1And L
2Can connect together, become above-mentioned L
1Defined joint, wherein, q is the integer of 2-6, perhaps m is the occasion beyond 0, L
2For-O-or-O-CH
2CH
2-O; L
3Be atomic bond or-(CH
2) r-, wherein r can be the integer of 1-6.
These polymkeric substance can use for example by the disclosed WO96/32434 of the part inventor (or United States Patent (USP) the 5th, 037, No. 969), WO96/33233 (or United States Patent (USP) the 5th, 925, No. 720) and WO97/06202 (or United States Patent (USP) the 5th, 929, No. 177) and Jo etc., Biomaterials, 21,605-612, the polymkeric substance of 2002 records itself are perhaps through some modifications or according to the polymkeric substance of its preparation.
According to the present invention, the X of above-mentioned general formula (I) is if saccharide residue, for example constitute the pair cell surface receptor part in conjunction with the monose in territory or oligosaccharides (for example, sugar unit can reach 11, preferably reach 7), particularly during the residue of disaccharides, the surface that is formed by related polymer can effectively prevent said culturing cell bonding among the present invention, so preferred especially.Represent to be documented in the polymkeric substance of monose among the above-mentioned WO96/32434 (or United States Patent (USP) the 5th, 037,969) about X, in addition,, can support required various saccharide residues according to the method for record wherein.Equally, X represents that the polymkeric substance of polymkeric substance, the expression oligosaccharides residue of polysaccharide residue radical can change and prepares by aforesaid method being done some.But, as another kind of method, with above-mentioned WO96/33233 (or United States Patent (USP) the 5th, 925, No. 720) after the X of the record polymkeric substance that partly has an acetal radical is converted into aldehyde radical, as required, amino is imported in monose or the oligosaccharides in advance, utilize this amino and above-mentioned aldehyde radical,, can obtain the polymkeric substance that X is a saccharide residue by the reductive amination reaction.As this monose or oligosaccharides, preferably contain 1 galactopyranose base at least.Unqualified to this oligosaccharides, as this oligosaccharides, can exemplify lactose, various saliva oligosaccharides.In addition, the X of above-mentioned general formula (I) is that the hydrophilic polymer of oligomeric peptide residue that constitutes the ligand binding domain of cell surface receptor also can preferably use in the present invention.This part can be the water-soluble signaling molecule of lectin (for example proteohormone, a growth factor protein matter).Unqualified to oligomeric peptide, as oligomeric peptide, can exemplify and contain arginine (Arg), glycine (Gly), aspartic acid (Asp) at least, and as a whole can be for water miscible, amino-acid residue reaches 10 oligomeric peptide.
Except above-mentioned and carbohydrate are covalently bound is the sugar derivatives of polymer based with PEG, polysaccharide can also be used as hydrophilic polymer by bonded polysaccharide itself as poly-semi-lactosi, sialic acid, other lectin in the present invention.For hydrophilic polymer, so long as those skilled in the art, can understand according to above-mentioned illustrational polymkeric substance, but under cell culture condition of the present invention, being meant water soluble under whole polymkeric substance condition around, is water miscible with the segment corresponding polymer (is polyethylene with above-mentioned example) that constitutes polymkeric substance perhaps.So long as those skilled in the art, reference embodiment described later, the just concrete polymkeric substance that can easily can utilize well in the present invention from these polymkeric substance selections.
Y in the general formula (I) according to circumstances is Y-L sometimes
1-(B)-, can according to desire form this hydroaropic substance surface lower floor's (can be directly or indirectly form substrate, the film of cultivating support surface, apparatus surface, film or vapor-deposited film) character and suitably select.For example, this support surface can have the polymkeric substance that makes general formula (I) and can adhere to securely or the bonded surface.Method to the surface that forms hydroaropic substance is unqualified, in this support surface coated siloxanes, perhaps the occasion that hydrophobizations such as silane treatment are handled has been implemented on the surface of for example containing hydroxyl, select part Y-L
1-(B), the m that makes it to become in the general formula (I) represents beyond 0 that for example the segmented copolymer of the integer more than 5 utilizes this ester segmental hydrophobicity, with this polymkeric substance attached to this support surface, thereby form the surface of hydroaropic substance.In such example, strengthen in order to make adhering to more of polymkeric substance, will with the corresponding homopolymer of this ester segment in advance attached to the support surface, and then this segmented copolymer is adhered to, then can make the hydrophilic substance surface that shows good resistance to peeling off etc.
In addition, the support surface has above-mentioned United States Patent (USP) the 5th, 976, the occasion of the functional group of No. 826 specification sheets records, group Y can be can with this functional group reactions, and form covalent linkage, for example-CONH-,-CONHCO-,-S-S-,-O-,-Si-O-,-other functional groups of NH-etc.The polymkeric substance of these functional groups being introduced the method for Y and containing these functional groups is documented in above-mentioned by in the disclosed patent specification of the part inventor.On the other hand, in No. the 5th, 976,826, above-mentioned United States Patent (USP), put down in writing a part about the content on the support surface with functional group is provided; In addition; Cement Composite Treated by Plasma is carried out according to itself known method in surface by will being coated with polymeric amide, urethane, polyacrylamide etc.; perhaps will have with the monomer of corresponding protected functional group of above-mentioned functional group and handle according to itself known Plasma Polymerization; then; as required protecting group is sloughed, can prepare.
In addition, when the support surface is formed by metals such as gold and silver, copper, be sulfydryl by making group Y, the so-called chemisorption of polymkeric substance that can make general formula (I) is made required surface in lower floor.
On the other hand, this zonule also can be handled as required, makes its surface become cellular affinity.Said in this specification sheets " cellular affinity " is meant when cultivating above-mentioned endotheliocyte, epithelial cell or inoblast on having the surface of this character, the culturing cell bonding, and the agglutinating cell washs or does the wash the surface that can not come off through demulcent like this.This surface can be by the change with hydrophobic group (alkyl, alkyl silyl, fluoro-alkyl etc.) contain thing or have charged group (for example ,-COO-,-PO
3H-etc.) surface that compound (comprising the protein that constitutes extracellular matrix) forms.The preparation on this surface can be carried out according to following method: in the making method on the surface of above-mentioned hydroaropic substance, replace hydroaropic substance (or hydrophilic polymer) to handle with above-mentioned any compound.
The zonule on cellular affinity surface can make according to well-known pattern forming method of those skilled in the art or micro-patterning method (micropatterning).But, can be preferably make according to following method, at first, on the support that is fit to, form the surface of hydrophilic polymer, be provided with in the above corresponding to the hole of the zonule pattern arranged of form as required, by this hole, for example use H
2+ N
2Cement Composite Treated by Plasma, remove and form this surperficial polymer layer, as required, with the above-mentioned zonule that can form the such gained of compound treatment on cellular affinity surface.What above-mentioned " as required " recorded and narrated is, so long as make the surface (can be the surface of device itself) of hydrophilic polymer surface deposition can not be subjected to the detrimentally affect that above-mentioned Cement Composite Treated by Plasma brings and the cellular affinity surface that forms, just there is no need to carry out above-mentionedly to handle arbitrarily.
Interval between the zonule of Zhi Zuoing is preferably the shortest like this is about 100 μ m.Selecting endotheliocyte or inoblast etc. to form in the culture system of cell as support dependent cell or feeder layer, if with this interval, surf zone by hydrophilic polymer is isolated a plurality of zonules, cultivation endotheliocyte on the then above-mentioned zonule or inoblast and cultivation parenchyma, particularly the liver cell spherule can not take place in fact to connect or build bridge each other.So-called " can not take place in fact to connect or build bridge " is meant, exist in most zonules the cultivation liver cell spherule on 1 zonule to be lower than 10% on form with the situation that cultivation liver cell spherule on other zonules is connected, preferably be lower than 5%, more preferably 0%.
Like this, (or patterning) of arranging by desired mode, the culturing cell that supports the coculture that comprises the spherule that culturing cell forms constitute thing, can be according to another embodiment of the present invention, promptly the preparation method of culturing cell formation thing obtains well.
(A) on the support surface of answering culturing cell, spin coating is a polymer based with the PEG fragment, polymkeric substance shown in the preferred above-mentioned general formula (I), X is organic solvent (for example toluene) solution or the aqueous solution of the polymkeric substance of saccharide residue or oligomeric peptide residue in the special preferred formula.In addition, for example the material on this support surface is the occasion of glass, handles if in advance its lip-deep hydroxyl is carried out hydrophobization with hydrophobic silane coupling agent, there is no need sometimes subsequently that then cellular affinity is carried out in the zonule and handles.Silane treatment can be with reference to the currently known methods of itself, H.O tsuka etc. for example, Biomacromolecules, 2000,1, the method for 21-27 record.In addition; polymkeric substance when also the corresponding group of X is acetal in spin coating in advance and the general formula (I); then, carry out the reductive amination reaction between the aldehyde that the amino that imported in advance in the amino sugar and the deprotection by above-mentioned acetal are exposed, thereby saccharide residue is introduced in the above-mentioned polymkeric substance.Kind according to the polymkeric substance that uses, the suitableeest thickness of this polymer layer changes, therefore can not limit the thickness of this polymer layer, as long as its thickness can prevent raising culturing cell on the zonule or the cell different with this culturing cell, for example cultivate parenchyma, and bond between these cells on other zonules and get final product.With regard to this thickness, those skilled in the art can easily determine by little experiment.Though unqualified to thickness, minimum must be the thickness of general unimolecular film.
(B) on the surface of step (A) gained, placement has the covering figure in hole, a plurality of zonules (toroidal of the preferred about 50-500 μ of diameter m), by plasma producing apparatus, uses
H
2+ N
2Carry out Cement Composite Treated by Plasma, destroy and remove polymer layer, expose the hydrophobicity treat surface corresponding to the zone in hole.
(C) in case of necessity, also can remove on the surface that polymer layer exposes, by above-mentioned covering figure, make monomer with cellular affinity group etc. carry out plasma polymerization or coating derives from this polymer of monomers, thereby this finishing is become cellular affinity.
(D) on the surface that has the cellular affinity zonule like this, for example endotheliocyte is (as long as suitable with cultivating, available commercially available cell, " the human umbilical vein's vascular endothelial cell " of big Japanese pharmaceutical manufacturing for example) substratum is (as long as suitable, available commercially available substratum, " the VE substratum " of big Japanese pharmaceutical manufacturing for example) cultivate these cells, form the culturing cell layer.This cultivation can be according to the method for for example speciallyying permit the record of No. 2973976 communique, or it is changed carries out.
(E) on the raising culturing cell layer that step (D) forms, cultivate and the different cell of cell that forms this cellular layer preferred parenchyma.Preferably first with the cell headed by the parenchyma for cell, but when cultivating according to method of the present invention, as long as can on above-mentioned cellular layer, form the culturing cell spherule, also can be that cell or any proterties of systematize (or strainization) transforms or cells transfected.In addition, the endotheliocyte or the inoblast that form feeder layer also can be used the cell that forms spherule, preferred parenchyma, and be not subjected to the restriction in its source, but preferably use zooblast of the same race.In this cell, the cultivation of cell also can according to itself known cultural method (for example, with reference to S.N.Bhatia etc., Biotech.Prog.1998,14,378-387) cultivate.Cultivation when using liver cell as this cell can form on the raising culturing cell layer on the above-mentioned zonule after 24 hours usually cultivate the liver cell spherule at least.
Other cell beyond the liver cell also can be cultivated according to itself known cultural method, forms the spherule of cultivating parenchyma on the raising culturing cell layer on the above-mentioned zonule.The cultural method of pair cell is unqualified, about myocardial cell's cultivation, and can be with reference to T.Shimizu etc., Journal of Biomedical Material Research, 60 (1) (2002): 110-117; G.Illiano etc., American Journal ofHypertension 15 (2002): 638-643; About the cultivation of spongiocyte, can be with reference to C.Gamboa etc., Neurochemistry International 40 (2002): 397-403; In addition, about the cultivation of pancreas β cell, can be with reference to J.L.Petit-Thevenin etc., Biochemica et Biophysica Acta 1530 (2001): 184-198.
The zooblast that uses among the present invention can be according to currently known methods separately, and for example surgical method obtains respectively, and is preferred just for cell, but also can use for example by (wealth) Humanscience development financial group and the commercially available cell of other suppliers.
The coculture that can access can be used to make with the culturing cell that has on the support surface and constitutes thing itself as surperficial biological device as mentioned above, perhaps forms surperficial biological device by this coculture.Unqualified to this biological device, as this biological device, can exemplify and be used to check the toxic device of cultivating parenchyma, be used to screen the infull medical treatment support of device, the parenchyma of material of activation culture parenchyma function with device and the device that is used to carry out parenchyma physiological action simulation test.
According to the present invention, the coculture that supports on the biological device (comprises the spherule that is formed by culturing cell, and the lower floor of the formation that comes down to individual layer and this spherule contact and with the different culturing cell of cell that forms this spherule) can remain on this apparatus surface steadily in the long term, and the exceptional function that keeps each parenchyma (for example, under the situation of the pancreas β cell beyond the liver cell, be the secretion of insulin function; Under myocardial cell's the situation, be the motor function of beating).For example, with the biological device of the present invention that liver cell can prepare, be the biological device that supports the uniform spherule of form with the regulation Pareto diagram, in addition, this spherule each zonule in fact has independently shape.And, the minimum high-caliber hepatocyte function (for example high-caliber albumin produces ability) that also can 3 weeks of stable maintenance of the spherule on the biological device of the present invention.Therefore, this biological device for example can be used to screen the environment or the material that can influence liver function.This influence can be by the variation and the product of monitoring spherule form, and the variation that produces ability as albumin is estimated.
According to the present invention, above-mentioned culturing cell constitutes coculture contained in the thing by physics or biochemical processing, and the culture of peeling off from support itself can be provided.The coculture of this free form also is a kind of mode of the present invention, for example, can be used for transplanting medical treatment, neomorph engineering, hybridization build artificial organs.
Below, in conjunction with specific examples, further specify the present invention, but the purpose that provides these examples is only for the ease of understanding the present invention.
A) making of cell cultures bed
With white slide glass (26 * 76 * 0.8mm/Takahashi Giken GlassCo., Ltd.) boiled 60 minutes with sulfuric acid/hydrogen peroxide (50/50), then clean, use 2%[3-(methacryloxy) propane in the ethanol/water (95/5) then] Trimethoxy silane solution, carry out silane coupledly, above-mentioned glass surface is carried out hydrophobic treatment.On the hydrophobic treatment slide surface of preparation like this, the spin coating molecular weight is 4% toluene solution of 20000 polylactide, then further spin coating (2% toluene solution of the lactose-PEG/PLA that obtains hereinafter referred to as acetal-hydroformylation thing PEG/PLA) and the reductive amination of aminophenyl lactose forms the thick macromolecule layer surface of about 100 μ m by acetal-polyoxyethylene glycol (molecular weight 6000)-copolymerization lactide (molecular weight 8000).On the macromolecule layer surface that obtains like this, place covering pattern, carry out H with the 100 μ m circular ports that separate at interval with 100 μ m
2+ N
2Cement Composite Treated by Plasma [ICPpower:500W, Bias power:30W (Vdc=60V), N
2+ H
2=50sccm/30sccm, 2 * 10-5Torr].By Cement Composite Treated by Plasma, form the hole (with reference to Fig. 1) of exposing glass surface according to above-mentioned covering figure ammonia.
On above-mentioned surface, apply the improved Eagle substratum (DMEN of Dulbecco, Gibco) (supplementation with insulin, foetal calf serum, hyperglycemic-glycogenolytic factor, the epitheliosis factor, penicillin, hydrocortisone and Streptomycin sulphate) forms the cell bond regions (or cell cultures bed) corresponding with above-mentioned hole.
B) cell cultures
On the cell cultures bed that a) obtains, behind 1 * 106cell/cm2 inoculation vascular endothelial cell (Bovine aorti endotherial cell), at 5%CO
2In the environment, under 37 ℃, leave standstill and cultivated 24 hours.Like this, endotheliocyte is just along the glass pattern in the hole of exposing bonding (with reference to Fig. 2) in the above.Subsequently, will by rat liver by collagenase perfusion method preparation first for cell by 1 * 106cell/cm2 inoculation after, at 5%CO
2In the environment, under 37 ℃, leave standstill and cultivated 24 hours, liver cell only is bonded in above-mentioned pattern-like and forms on the endotheliocyte in zone, forms globular array (with reference to Fig. 3).The spherical physical efficiency of these livers is kept liver function (for example albumin generation ability) at least 3 weeks, can confirm cytoskeleton (about 3 dimension enlarged views of spherule, with reference to Fig. 5).
In addition, on lactose-PEG/PLA surface, when the interval of the circular port of 100 μ m was lower than 100 μ m, the pattern of endotheliocyte and the cell of adjacency connected together, if further shorten at interval, cell can become sheet (with reference to Fig. 4) sometimes.Usually, if there is no the endothelial layer of patterning then can not form and cultivate hepatocellular spherule.
Industrial applicibility
In for example liver orbicule array that obtains as mentioned above, the spherical physical efficiency long term maintenance of these livers Liver function, and can there be for example tens thousand of liver orbicules in each culture dish (in theory Without limits), therefore can be used for tens thousand of kinds medicine calibrating for 1 time. In addition, by this array The coculture of peeling off is for example cultivated the liver orbicule, can be used for transplanting medical treatment, liver regeneration worker The technical field of Cheng Xue. Therefore, the present invention can be applied to check for estimating drug effect Industry or medical treatment support to use industry.
Claims (25)
1. a culturing cell constitutes thing, be to constitute thing at the culturing cell of coculture more than a kind or 2 kinds that contains different mutually zooblasts on the support, wherein, comprise coculture and support, described coculture comprises: origin come from a kind should the difference cell the thin spherule that forms of cultivation, and the individual layer that exists on the zonule of support, described individual layer forms the lower floor that contact with this spherule, and comes down to the individual layer of culturing cell formation that origin comes from the another kind of cell of the cells survival that can make this spherule of formation and/or performance function.
2. culturing cell according to claim 1 constitutes thing, and wherein, the cell that forms spherule is a parenchyma.
3. culturing cell according to claim 1 constitutes thing, wherein, the cell that forms spherule is the parenchyma that is selected from liver cell, pancreas β cell, myocardial cell, spongiocyte, skin epithelial cell, chondrocyte, osteocyte and stem cell, and in addition a kind cell different with the cell that forms this spherule is selected from endotheliocyte, inoblast and epithelial cell.
4. culturing cell according to claim 1 constitutes thing, and wherein, the cell that forms spherule is a liver cell, and in addition a kind culturing cells different with the cell that forms this spherule derive from endotheliocyte or inoblast.
5. culturing cell according to claim 1 constitutes thing, wherein, has coculture more than 2 kinds, and exists in mutual isolated mode.
6. culturing cell according to claim 1 constitutes thing, and wherein, the zonule has the area of about 1500-30000 μ m2.
7. culturing cell according to claim 1 constitutes thing, and wherein, the zonule is the substantial circle of the about 50-500 μ of diameter m.
8. culturing cell according to claim 1 constitutes thing, wherein, has coculture more than 2 kinds, and the zonule is the substantial circle of the about 50-200 μ of diameter m, and the shortest between the zonule of each adjacency is spaced apart about 100 μ m.
9. culturing cell according to claim 1 constitutes thing, wherein, has coculture more than 2 kinds, and the zonule that supports each culture separates by the support surface of being made by the material of wetting ability and the non-affinity of cell.
10. culturing cell according to claim 1 constitutes thing, and wherein, the material of wetting ability and the non-affinity of cell is to be in the polymer based with the polyoxyethylene glycol segment, and any one end of this segmental has the polymkeric substance of hydroxyl.
11. culturing cell according to claim 1 constitutes thing, wherein, the material of wetting ability and the non-affinity of cell has polyoxyethylene glycol segment and polylactide segment.
12. culturing cell according to claim 1 constitutes thing, wherein, the material of wetting ability and the non-affinity of cell is selected from based on the derivative of polyoxyethylene glycol segment and polysaccharide, any one terminal covalent attachment of this polyoxyethylene glycol segment from the part that constitutes cell surface receptor in conjunction with the compound of selecting the monose in territory or oligosaccharides and the oligomeric peptide.
13. culturing cell according to claim 1 constitutes thing, wherein, the material of wetting ability and the non-affinity of cell is the sugar derivatives based on the polyoxyethylene glycol segment, any one terminal covalent attachment of this polyoxyethylene glycol segment contain the monose or the oligosaccharides of at least 1 galactosyl.
14. culturing cell according to claim 1 constitutes thing, wherein, the material of wetting ability and the non-affinity of cell has been included in any one terminal covalent attachment and has contained the monose of at least 1 galactopyranose base or the polyoxyethylene glycol segment and the polylactide segmental sugar derivatives of oligosaccharides.
15. culturing cell according to claim 1 constitutes thing, wherein, the material of wetting ability and the non-affinity of cell is the peptide derivant based on the polyoxyethylene glycol segment, any one terminal covalent attachment of this polyoxyethylene glycol segment contain the oligomeric peptide of the aminoacid sequence of arginine-glycine-aspartic acid.
16. culturing cell according to claim 1 constitutes thing, wherein, the zonule has the surface made from the cellular affinity material.
17. culturing cell according to claim 1 constitutes thing, wherein, the zonule has the surface made from the cellular affinity material, this cellular affinity material be selected from have alkyl, the compound of alkyl silyl and fluoro-alkyl; Compound with carboxylate anion and phosphate radical anion; And the protein that constitutes extracellular matrix.
18. a coculture is the coculture of being peeled off by the support that any described culturing cell among the claim 1-17 constitutes thing.
19. a biological device comprises by any described culturing cell among the claim 1-17 and constitutes the surface that thing forms.
20. biological device according to claim 19, wherein, culturing cell constitutes the sphere that each spherule of coculture more than 2 kinds in the thing comes down to the about 50-100 μ of diameter m, and the shortest between the spherule of each spherule and each adjacency is spaced apart about 100 μ m, and is arranged in certain pattern.
21. biological device according to claim 19, this biological device be used to check the Cytotoxic device that forms spherule, be used to screen the material of the cell function that activation forms spherule device, form the infull medical treatment support of the cell of spherule with device and the device that is used to carry out the simulation test of parenchyma physiological action.
22. the culturing cell of coculture more than a kind or 2 kinds that contains different mutually zooblasts on the support constitutes the preparation method of thing, this culturing cell constitutes thing and is included in the coculture that supports the spherule that is formed by culturing cell on a plurality of segregate zonules, it is characterized in that, on the material surface of coculture that should bond, formation is the polymer based layer with the polyoxyethylene glycol segment, any one terminal covalent attachment of this polyoxyethylene glycol segment or not covalent attachment from the compound of selecting in conjunction with the monose in territory or oligosaccharides and oligomeric peptide of the part that constitutes the zooblast surface receptor, by having the covering pattern of a plurality of intervals at least about the circular port of the about 50-100 μ of the diameter of 100 μ m m, above-mentioned polymer layer is carried out Cement Composite Treated by Plasma, thereby remove polymer layer with above-mentioned hole respective regions, as required, coating cellular affinity material on the zonule of having removed polymer layer, on this zonule, cultivate endotheliocyte or inoblast, form the individual layer of culturing cell, on this culturing cell, cultivate the cell different then, form the spherule of this difference cell with this culturing cell.
23. culturing cell according to claim 22 constitutes the preparation method of thing, wherein, the cell of formation spherule is selected from liver cell, pancreas β cell, myocardial cell, spongiocyte, skin epithelial cell, chondrocyte, osteocyte and embryo or adult stem cell.
24. culturing cell according to claim 22 constitutes the preparation method of thing, wherein, the cell that forms spherule is a liver cell.
25. culturing cell according to claim 22 constitutes the preparation method of thing, wherein, polymer layer by in any one terminal covalent attachment monose or oligosaccharides be that polymer based constitutes with the polyoxyethylene glycol segment.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001226293 | 2001-07-26 | ||
| JP226293/2001 | 2001-07-26 | ||
| PCT/JP2002/007539 WO2003010302A1 (en) | 2001-07-26 | 2002-07-25 | Cultured cell construct containing spheroids of cultured animal cells and utilization thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1798832A true CN1798832A (en) | 2006-07-05 |
| CN1798832B CN1798832B (en) | 2010-04-28 |
Family
ID=19059133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN028148428A Expired - Fee Related CN1798832B (en) | 2001-07-26 | 2002-07-25 | Cultured cell construct containing spheroids of cultured animal cells and utilization thereof |
Country Status (9)
| Country | Link |
|---|---|
| US (2) | US7470424B2 (en) |
| EP (2) | EP1428871B1 (en) |
| JP (1) | JP4111446B2 (en) |
| KR (1) | KR100675533B1 (en) |
| CN (1) | CN1798832B (en) |
| AT (1) | ATE406436T1 (en) |
| CA (1) | CA2454807C (en) |
| DE (2) | DE60236974D1 (en) |
| WO (1) | WO2003010302A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955595A (en) * | 2010-08-11 | 2011-01-26 | 东南大学 | Method for guiding fixed-point cell growth by preparing chemical micro-patterns on surfaces of various materials |
| CN104894051A (en) * | 2015-05-27 | 2015-09-09 | 成都易创思生物科技有限公司 | Novel cell culture method |
| CN106133503A (en) * | 2014-03-26 | 2016-11-16 | 株式会社斯库林集团 | Spheroid evaluation methodology and spheroid evaluating apparatus |
Families Citing this family (52)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2461715A1 (en) * | 2001-10-02 | 2003-04-10 | Becton, Dickinson And Company | Proliferation and differentiation of stem cells using extracellular matrix and other molecules |
| US20060234377A1 (en) | 2003-02-06 | 2006-10-19 | Teruo Okano | Cell sheets for ectocornea formation, method of producing the same and method of using the same |
| AU2003207288A1 (en) * | 2003-02-06 | 2004-08-30 | Cellseed Inc. | Anterior ocular-associated cell sheet, three-dimensional construct and process for producing the same |
| WO2004069988A1 (en) * | 2003-02-06 | 2004-08-19 | Cellseed Inc. | High-density cell array board, process for producing the same and method of using the same |
| JP4649935B2 (en) * | 2004-02-02 | 2011-03-16 | コニカミノルタホールディングス株式会社 | Inkjet printer |
| WO2005081970A2 (en) | 2004-02-24 | 2005-09-09 | The Curators Of The University Of Missouri | Self-assembling cell aggregates and methods of making engineered tissue using the same |
| EP1859817A4 (en) | 2005-02-28 | 2012-11-07 | Cellseed Inc | Cultured cell sheet, process for producing the same and method for restoring tissue using the same |
| EP1857126B1 (en) * | 2005-02-28 | 2018-10-24 | CellSeed Inc. | Cultured cell sheet, production method thereof, and application method thereof |
| JP2008545410A (en) * | 2005-05-24 | 2008-12-18 | ザ レジェンツ オブ ザ ユニヴァースティ オブ カリフォルニア | Microscale micropatterned in vitro manipulation / fabrication organization |
| CA2568475C (en) * | 2005-11-18 | 2016-08-30 | Lifescan, Inc. | A method for creating cell clusters |
| AU2013202489B2 (en) * | 2005-11-18 | 2016-03-03 | Lifescan, Inc. | A method for creating cell clusters |
| JP5070565B2 (en) | 2006-05-29 | 2012-11-14 | 大日本印刷株式会社 | Cell culture substrate |
| US20090203536A1 (en) * | 2006-06-06 | 2009-08-13 | Vermette Patrick | Assay supports comprising a peg support, said support attached from a peg solution in cloud point (theta solvent) conditions |
| JP5261920B2 (en) | 2006-11-10 | 2013-08-14 | 大日本印刷株式会社 | Test methods and test kits using cells |
| US8592139B2 (en) | 2006-11-10 | 2013-11-26 | Dai Nippon Printing Co., Ltd. | Test method using cells and test kit therefor |
| EP2120546B1 (en) * | 2006-12-18 | 2016-09-28 | Ben Gurion University Of The Negev | Scaffolding for tissue regeneration or repair |
| JP5583312B2 (en) | 2007-02-20 | 2014-09-03 | 富士フイルム株式会社 | Tissue formation substrate, tissue formation kit, tissue formation method using the same, and three-dimensional tissue formed by the tissue formation method |
| WO2009050965A1 (en) * | 2007-10-19 | 2009-04-23 | Japan As Represented By General Director Of Agency Of National Center For Child Health And Development | Support for immobilizing frozen cells, tool for primary hepatocyte culture and method of producing tool for primary hepatocyte culture |
| JP5388126B2 (en) * | 2007-12-06 | 2014-01-15 | 学校法人東京理科大学 | Branched polyalkylene glycol derivative, photosensitive composition, crosslinked product and substrate |
| CN102124331B (en) | 2008-06-24 | 2013-10-30 | 密苏里大学 | Self-assembling multicellular bodies and methods of producing three-dimensional biological structure using same |
| KR101005874B1 (en) * | 2008-08-27 | 2011-01-06 | 한길토건(주) | Construction method of falling rock depense wall which is available for steel beam |
| US9598668B2 (en) * | 2008-10-14 | 2017-03-21 | Cellseed Inc. | Temperature-responsive cell culture substrate and method for producing the same |
| JP5496488B2 (en) * | 2008-10-22 | 2014-05-21 | 学校法人東京女子医科大学 | Cell pattern collection tool |
| EP2358860A1 (en) * | 2008-11-21 | 2011-08-24 | Corning Incorporated | Spaced projection substrates and devices for cell culture |
| US20100166822A1 (en) * | 2008-12-31 | 2010-07-01 | Howmedica Osteonics Corp. | Adhesive cartilage implant |
| JP2010233538A (en) * | 2009-03-31 | 2010-10-21 | Trans Parent:Kk | Method for evaluating substrate metabolite |
| JP2012523238A (en) | 2009-04-09 | 2012-10-04 | ジ・アリゾナ・ボード・オブ・リージェンツ・オン・ビハーフ・オブ・ザ・ユニバーシティ・オブ・アリゾナ | Cell seeding and co-culture of 3D fibroblast constructs |
| HUP0900819A2 (en) * | 2009-05-05 | 2011-01-28 | Pecsi Tudomanyegyetem | Lung tissue culture |
| US8481303B2 (en) | 2009-10-12 | 2013-07-09 | Corning Incorporated | Microfluidic device for cell culture |
| WO2011110722A1 (en) * | 2010-03-12 | 2011-09-15 | Fundación Progreso Y Salud | Method for the in vitro proliferation of cells from endodermal tissues |
| WO2012048170A2 (en) * | 2010-10-06 | 2012-04-12 | Massachusetts Institute Of Technology | Humanized animals via tissue engineering and uses therefor |
| CA2841902A1 (en) | 2011-07-25 | 2013-01-31 | Cytoo | Methods and a device for the formation of three-dimensional multicellular assemblies |
| CA2878404A1 (en) | 2012-07-10 | 2014-01-16 | The Trustees Of The University Of Pennsylvania | Biomaterials for enhanced implant-host integration |
| EP2902496B1 (en) | 2012-09-27 | 2018-04-18 | Kuraray Co., Ltd. | Method for evaluating effect of cytokine on metabolic activity of cytochrome p450, and drug screening method |
| WO2014131019A2 (en) | 2013-02-25 | 2014-08-28 | Ohio State Innovation Foundation | Her-1, her-3 and igf-1r compositions and uses thereof |
| WO2014143998A1 (en) * | 2013-03-15 | 2014-09-18 | The Broad Institute | Systems and methods for culturing epithelial cells |
| US9790465B2 (en) | 2013-04-30 | 2017-10-17 | Corning Incorporated | Spheroid cell culture well article and methods thereof |
| KR101647793B1 (en) * | 2014-09-02 | 2016-08-12 | 충남대학교산학협력단 | Cell culture container for formation spheroid using thermosensitive glycol chitosan derivatives and spheroid formation method using the same |
| WO2016069895A1 (en) | 2014-10-29 | 2016-05-06 | Corning Incorporated | Cell culture insert |
| CN107109339B (en) | 2014-10-29 | 2021-07-02 | 康宁股份有限公司 | Perfusion bioreactor platform |
| CN104630148B (en) * | 2015-01-19 | 2017-06-06 | 西安交通大学 | A kind of cell ball in-situ preparation method based on hydrogel microwell plate |
| WO2017062757A1 (en) | 2015-10-08 | 2017-04-13 | Massachusetts Institute Of Technology | In situ expansion of engineered devices for regeneration |
| JP7033066B2 (en) | 2016-01-07 | 2022-03-09 | ビー．ジー．ネゲブ テクノロジーズ アンド アプリケーションズ リミテッド， アット ベン‐グリオン ユニバーシティー | Compositions and Methods to Generate Immune Tolerance Responses |
| WO2018066953A1 (en) * | 2016-10-07 | 2018-04-12 | 울산과학기술원 | Hanging drop cell culture device using porous membrane, method for manufacturing same, hanging drop cell culture method using same, and hanging drop cell culture automation device |
| WO2019014635A1 (en) | 2017-07-14 | 2019-01-17 | Corning Incorporated | 3d cell culture vessels for manual or automatic media exchange |
| US11584906B2 (en) | 2017-07-14 | 2023-02-21 | Corning Incorporated | Cell culture vessel for 3D culture and methods of culturing 3D cells |
| EP3652291B1 (en) | 2017-07-14 | 2021-12-29 | Corning Incorporated | Cell culture vessel |
| US11857970B2 (en) | 2017-07-14 | 2024-01-02 | Corning Incorporated | Cell culture vessel |
| PL3649226T3 (en) | 2018-07-13 | 2022-05-16 | Corning Incorporated | Microcavity dishes with sidewall including liquid medium delivery surface |
| CN111065725B (en) | 2018-07-13 | 2024-03-29 | 康宁股份有限公司 | Fluidic device comprising microplates with interconnected walls |
| EP3649229B1 (en) | 2018-07-13 | 2021-08-25 | Corning Incorporated | Cell culture vessels with stabilizer devices |
| KR20210070648A (en) * | 2019-12-05 | 2021-06-15 | 한국화학연구원 | cardiomyocyte spheroid formed on cell nonadhesive PEG polymer substrate and method for preparing the same |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2545100B1 (en) * | 1983-04-29 | 1985-12-20 | Inst Nat Sante Rech Med | PROCESS FOR OBTAINING CULTURES OF HUMAN HEPATOCYTES, THE CULTURES OBTAINED AND THEIR BIOLOGICAL AND BIOCHEMICAL APPLICATIONS |
| US5037969A (en) | 1986-07-03 | 1991-08-06 | Takeda Chemical Industries, Ltd. | Glycosyl derivatives and use thereof |
| JPH0751061B2 (en) | 1989-06-03 | 1995-06-05 | 鐘淵化学工業株式会社 | Manufacturing method of cell array control tool |
| DE3938632C1 (en) * | 1989-11-21 | 1991-03-14 | Adelbert Prof. Dr.Dr. 8046 Garching De Bacher | Culturing living animal cells - using natural or synthetic polymer substrate vacuum deposition coated with e.g. nitride of Gp=III element |
| EP0529751A1 (en) * | 1991-08-09 | 1993-03-03 | W.R. Grace & Co.-Conn. | Cell culture substrate, test material for cell culture and preparations thereof |
| US5776748A (en) * | 1993-10-04 | 1998-07-07 | President And Fellows Of Harvard College | Method of formation of microstamped patterns on plates for adhesion of cells and other biological materials, devices and uses therefor |
| JP2609073B2 (en) | 1994-10-26 | 1997-05-14 | 鐘淵化学工業株式会社 | Cell arrangement control tool and cell arrangement control method |
| EP0852243B1 (en) | 1995-04-14 | 2002-10-30 | Kazunori Kataoka | Polyethylene oxides having saccharide residue at one end and different functional group at another end, and process for producing the same |
| RU2169742C2 (en) | 1995-04-19 | 2001-06-27 | Катаока Казунори | Heterotelochelate block copolymer and method of preparation thereof |
| RU2174989C2 (en) | 1995-08-10 | 2001-10-20 | Катаока Казунори | Block copolymer having functional groups on both ends |
| JP2002510969A (en) * | 1997-05-14 | 2002-04-09 | ザ ジェネラル ホスピタル コーポレーション | Co-culture of cells in micropatterned morphology |
| JP3077628B2 (en) * | 1997-05-27 | 2000-08-14 | 日本電気株式会社 | Cytotoxicity test method |
| JP2973976B2 (en) | 1997-06-23 | 1999-11-08 | 日本電気株式会社 | Toxicity test method using adherent cells |
-
2002
- 2002-07-25 US US10/484,839 patent/US7470424B2/en not_active Expired - Fee Related
- 2002-07-25 EP EP02755656A patent/EP1428871B1/en not_active Expired - Lifetime
- 2002-07-25 WO PCT/JP2002/007539 patent/WO2003010302A1/en active IP Right Grant
- 2002-07-25 DE DE60236974T patent/DE60236974D1/en not_active Expired - Lifetime
- 2002-07-25 EP EP08005978A patent/EP1939280B1/en not_active Expired - Lifetime
- 2002-07-25 DE DE60228594T patent/DE60228594D1/en not_active Expired - Lifetime
- 2002-07-25 JP JP2003515653A patent/JP4111446B2/en not_active Expired - Fee Related
- 2002-07-25 CA CA2454807A patent/CA2454807C/en not_active Expired - Fee Related
- 2002-07-25 KR KR1020047000851A patent/KR100675533B1/en not_active IP Right Cessation
- 2002-07-25 AT AT02755656T patent/ATE406436T1/en not_active IP Right Cessation
- 2002-07-25 CN CN028148428A patent/CN1798832B/en not_active Expired - Fee Related
-
2008
- 2008-09-08 US US12/230,916 patent/US7691369B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955595A (en) * | 2010-08-11 | 2011-01-26 | 东南大学 | Method for guiding fixed-point cell growth by preparing chemical micro-patterns on surfaces of various materials |
| CN106133503A (en) * | 2014-03-26 | 2016-11-16 | 株式会社斯库林集团 | Spheroid evaluation methodology and spheroid evaluating apparatus |
| CN106133503B (en) * | 2014-03-26 | 2019-01-22 | 株式会社斯库林集团 | Orbicule evaluation method and orbicule evaluating apparatus |
| CN104894051A (en) * | 2015-05-27 | 2015-09-09 | 成都易创思生物科技有限公司 | Novel cell culture method |
Also Published As
| Publication number | Publication date |
|---|---|
| US20040197907A1 (en) | 2004-10-07 |
| DE60228594D1 (en) | 2008-10-09 |
| EP1939280A1 (en) | 2008-07-02 |
| JPWO2003010302A1 (en) | 2005-06-02 |
| US7691369B2 (en) | 2010-04-06 |
| US7470424B2 (en) | 2008-12-30 |
| EP1428871A1 (en) | 2004-06-16 |
| WO2003010302A1 (en) | 2003-02-06 |
| EP1939280B1 (en) | 2010-07-07 |
| CA2454807A1 (en) | 2003-02-06 |
| KR20040025700A (en) | 2004-03-24 |
| CN1798832B (en) | 2010-04-28 |
| US20090011504A1 (en) | 2009-01-08 |
| EP1428871B1 (en) | 2008-08-27 |
| DE60236974D1 (en) | 2010-08-19 |
| ATE406436T1 (en) | 2008-09-15 |
| EP1428871A4 (en) | 2004-11-24 |
| KR100675533B1 (en) | 2007-01-29 |
| JP4111446B2 (en) | 2008-07-02 |
| CA2454807C (en) | 2012-09-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1798832B (en) | Cultured cell construct containing spheroids of cultured animal cells and utilization thereof | |
| Tang et al. | Recent development of temperature-responsive surfaces and their application for cell sheet engineering | |
| US20070026518A1 (en) | Controlling stem cell destiny with tunable matrices | |
| WO2006093207A1 (en) | Base material for regulating the differentiation/proliferation of cells | |
| WO2004085606A1 (en) | Cell culture medium and solidified preparation of cell adhesion protein or peptide | |
| CN107916260B (en) | Hydrogel-based single cell wrapping method, product and application | |
| US8013121B2 (en) | Isolated nature-identical collagen | |
| US9546349B2 (en) | Supports for cell culture and cell sheet detachment and methods for cell sheet detachment | |
| US20190040353A1 (en) | Polymer-based material having covalently bonded enzymatically degradable peptide sequences | |
| JP4336756B2 (en) | Animal cell culture constructs | |
| JP5124125B2 (en) | Cell culture substrate and method for producing cell culture substrate | |
| Barhouse et al. | Home Away From Home: Bioengineering Advancements to Mimic the Developmental and Adult Stem Cell Niche | |
| Pahnke et al. | Spatial and electrical factors regulating cardiac regeneration and assembly | |
| KR102654516B1 (en) | Cell encapsulation layer, encapsulated cell, cell encapsulation composition and preparation method thereof | |
| WO2001079453A1 (en) | Cell culture method, cell cluster obtained by said methods and use thereof | |
| EP2725099B1 (en) | Use of cell culture membrane, cell culture substrate in cell culture | |
| JP2011130720A (en) | Cell culture substrate and method of using the same | |
| JP4381000B2 (en) | Animal cell culture material | |
| Farouz | Designing biomaterials for controlled cardiac stem cell differentiation and enhanced cell therapy in the treatment of congestive heart failure | |
| JPH02308790A (en) | Cell culturing method | |
| XUEJUN | Rapid Construction of Mechanically-Confined Multi-Cellular Structures Using Dendrimeric Inter-Cellular Linker | |
| Smith | Engineering Poly (ethylene glycol) Materials to Promote Cardiogenesis | |
| JPH05336956A (en) | Float culture of anchorage-dependent cell | |
| CN104818252A (en) | 3D (three-dimensional) cell culture material and application thereof | |
| JP2008283991A (en) | Method for reproducing cardiac diseased tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100428 Termination date: 20140725 |
|
| EXPY | Termination of patent right or utility model |